CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation

Abstract

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Full text

Feature Review
CRISPR/Cas-mediated plant genome editing:
outstanding challenges a decade after
implementation
Teodoro Cardi ,
1,2
Jana Murovec ,
3
Allah Bakhsh ,
4,5
Justyna Boniecka ,
6,7
Tobias Bruegmann ,
8
Simon E. Bull ,
9,10
Tom Eeckhaut ,
11
Matthias Fladung ,
8
Vladislava Galovic ,
12
Anna Linkiewicz ,
13
Tjasa Lukan ,
14
Isabel Mafra ,
15
Krzysztof Michalski ,
16
Musa Kavas ,
17
Alessandro Nicolia ,
2
Justyna Nowakowska ,
13
Laszlo Sági ,
18
Cecilia Sarmiento ,
19
Kubilay Yıldırım ,
17
Milica Zlatković,
12
Goetz Hensel ,
20,21
and Katrijn Van Laere
11,
*
The discovery of the CRISPR/Cas genome-editing system has revolutionized our
understanding of the plant genome. CRISPR/Cas has been used for over a de-
cade to modify plant genomes for the study of specific genes and biosynthetic
pathways as well as to speed up breeding in many plant species, including
both model and non-model crops. Although the CRISPR/Cas system is very effi-
cient for genome editing, many bottlenecks and challenges slow down further
improvement and applications. In this review we discuss the challenges that
can occur during tissue culture, transformation, regeneration, and mutant detec-
tion. We also review the opportunities provided by new CRISPR platforms and
specific applications related to gene regulation, abiotic and biotic stress re-
sponse improvement, and de novo domestication of plants.
A decade of CRISPR/Cas plant genome editing
Ensuring food security for a growing global population in a changing climate presents a great
challenge for agriculture. Conventional breeding can only tackle this challenge to some extent;
the classically obtained genetic gain will not suffice in the long term. Recent advances in genome
editing such as gene engineering using CRISPR/Cas (see Glossary) have opened many oppor-
tunities to accelerate plant breeding and to bridge the gap between conventional breeding and
the knowledge acquired through plant molecular biology to study and improve (complex) traits
[1]. CRISPR/Cas-mediated genome editing enables very precise and efficient targeted modifica-
tion in most crops, and thus largely increases the speed of crop improvement compared to con-
ventional breeding [2]. Since the first description of CRISPR/Cas as a plant genome-editing
technique, the technology has been successfully applied in close to 120 crops and model plants,
with reports of wide applications for as many as half of them (Table S1 in the supplemental infor-
mation online; www.eu-sage.eu/genome-search). CRISPR/Cas-edited plants are obtained by
mutagenesis techniques using site-directed nucleases (SDNs) which can introduce targeted
changes into specific DNA sequences of the genome to improve desired traits [3,4]. A distinction
can be made between site-directed nuclease type I (SDN-1),site-directed nuclease type
II (SDN-2), and site-directed nuclease type III (SDN-3) techniques because they result in dif-
ferent editing outcomes [5]. The CRISPR/Cas field is advancing very quickly (Figure 1, Key figure),
and CRISPR systems are undergoing continual modification to increase specificity. Base editing
(BE) and prime editing (PE) approaches do not rely on the repair of a double-strand break
(DSB) in DNA. The former induces C to T or A to G transitions through a deaminase fusedto either
Highlights
Climatechangeandthediversityofcon-
sumer needs require innovative methods
to continuously and rapidly modify
existing crops for the development of
new varieties.
In the past decade genome editing
by CRISPR/Cas and derivatives has
emerged as a novel and effective tech-
nology for functional studies and gene
discovery as well as for breeding new
traits and genotypes.
The development of novel CRISPR/Cas
platforms, methods for the delivery of
editing reagents, and methods for con-
trolling gene regulation and detection of
mutants have all expanded the scope
of genome editing and other CRISPR/
Cas-based approaches.
1
Consiglio Nazionale delle Ricerche
(CNR), Institute of Biosciences and
Bioresources (IBBR), Portici, Italy
2
CREA Research C entre for Veget able
and Ornamental Crops, Pontecagnano,
Italy
3
University of Ljubljana, Biot echnical
Faculty, Ljubljana, Slovenia
4
Department of Agricultural Genetic
Engineering, Faculty of Agricultural
Sciences and Technologies, Nigde Omer
Halisdemir University, Nigde, Turkey
5
Centre of Excellence in Molecular
Biology, University of the Punjab,
Lahore, Pakistan
6
Department of Genetics, Faculty of
Biological and Veterinary Sciences,
Nicolaus Copernicus University, Toruń,
Poland
Trends in Plant Science, Month 2023, Vol. xx, No. xx https://doi.org/10.1016/j.tplants.2023.05.012 1
© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Trends in
Plant Science OPEN ACCESS
TRPLSC 2472 No. of Pages 22

aCas nickase or a dead Cas (dCas), whereas the latter mediates targeted insertions, dele-
tions, and all base-to-base conversions by exploiting an engineered reverse transcriptase en-
zyme and a PE guide RNA (pegRNA) fused to a Cas nickase (reviewed in [6]). Furthermore,
new CRISPR/Cas delivery methods are being applied in addition to the classical transformation
system using Rhizobium radiobacter or Rhizobium rhizogenes (formerly Agrobacterium
tumefaciens and Agrobacterium rhizogenes, respectively). Some of these new technologies
make genome alteration possible without the use of recombinant DNA [3]. Despite these advan-
tages, many challenges remain regarding tissue culture and regeneration, mutant screening,
polyploidy, and multiplexing. This review lists the known challenges and presents the methods
and new platforms that are being developed to resolve some of these bottlenecks (Figure 1).
Tissue culture, transformation, and regeneration
Starting with the first attempts to deliver editing reagents through conventional transformation
procedures, novel delivery systems have been developed for different species to facilitate
in vitro tissue culture response, avoid stable transgene integration, test the functionality and effi-
ciency of editing constructs, and develop genotype-independent transformation/regeneration
protocols (Figure 2).
In vitro transformation and regeneration responses
Improvements in delivery methods and in fertile plant regeneration by Rhizobium-mediated trans-
formation have been recently achieved to overcome genotype-dependency. A ternary pVir sys-
tem, based on improved accessory plasmids characterized by small size, enhanced vector
stability, improved bacterial selectable marker, and amended vir genes, has resulted in more ef-
ficient T-DNA delivery and stable plant transformation in recalcitrant maize and sorghum varieties
[7–9]. Using this system, Che et al. [9] showed, however, that the main bottleneck in achieving
genotype-independent transformation of immature sorghum embryos is not the delivery step
but the tissue culture response for producing embryogenic callus.
The tissue culture response has recently been improved, for example, in Brassica napus [10], by
using epicotyl and higher stem (internodal) segments as explants, as well as in barley, where an
anther culture-based system was implemented [11]. These systems enable the development of
genotype-independent transformation and editing protocols.
Developmental regulators operate in concert with plant hormones in tissue culture to induce em-
bryogenesis or organogenesis from somatic cells [12]. The application of such regulators repre-
sents a recent improvement in tissue culture and transformation methods in various crops. In
several instances the transfer of genes encoding such regulators, including WUSCHEL
(WUS2), BABY BOOM (BBM), and SHOOT MERISTEMLESS (STM), has facilitated totipotency
[13]. However, the expression of these genes can cause genotype-specific pleiotropic effects
which prevent the recovery of normal, fertile plants. It is therefore important to modulate their ex-
pression, for instance by implementing inducible promoters, applying recombinase-mediated ex-
cision, or using cotransformation with two Rhizobium strains –one of which provides a T-DNA for
WUS2 expression and the other carries a T-DNA containing the gene of interest and a selectable
marker in a neighboring cell (a phenomenon designated 'altruistic transformation') [9,14].
The overexpression of a chimeric protein combining transcription factor GROWTH REGULATING
FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1), or Arabidopsis GRF5
and/or its homologs, also enhanced regeneration and transformation in monocots and dicots, in-
cluding woody species. Importantly, the GRF4–GIF1/GRF5 technology results in fertile and nor-
mal transgenic plants without the need for specialized promoters or transgene excision. This
7
Centre for Moder n Interdisciplinary
Technologies, Nicolaus Copernicus
University, Toruń, Poland
8
Thünen Institute of Forest Genetics,
Grosshansdorf, Germany
9
Molecular Plant Breeding, Institute of
Agricultural Sciences, Eidgenössische
Technische Hochschule (ETH) Zurich,
Switzerland
10
Plant Biochemistry, Institute of
Molecular Plant Biology, ETH, Zurich,
Switzerland
11
Flanders Research Institute for Agricul-
tural, Fisheries and Food, Melle, Belgium
12
University of No vi Sad, Institute of
Lowland Forestry and Environment
(ILFE), Novi Sad, Serbia
13
Molecular Biology and Genetics
Department, Institute of Biological
Sciences, Faculty of Biology and
Environmental Sciences, Cardinal Stefan
Wyszyński University, Warsaw, Poland
14
National Institute of Biology,
Department of Biotechnology and
Systems Biology, Ljubljana, Slovenia
15
Rede de Química e Tecnologia
(REQUIMTE) Labo ratório Associado
para a Química Verde (LAQV), Faculdade
de Farmácia, Universidade do Por to,
Porto, Portugal
16
Plant Breeding and Acclimatization
Institute, National Research Institute,
Błonie, Poland
17
Department of Molecular Biology and
Genetics, Faculty of Science, Ondokuz
Mayis University, Samsun, Turkey
18
Centre for Agricultural Resear ch,
Loránd Eötvös Research Network,
Martonvásár, Hungary
19
Department of Chemistry and
Biotechnology, Tallinn University of
Technology, Tal linn, Est onia
20
Heinrich-Heine-University, Institute of
Plant Biochemistry, Centre for Plant
Genome Engineering, Düsseldorf,
Germany
21
Division of Molecular Biology, Centre
of the Region Hana for Biotechnological
and Agriculture Rese arch, Fac ulty of
Science, Palacký University, Olomouc,
Czech Republic
*Correspondence:
katrijn.vanlaere@ilvo.vlaanderen.be
(K. Van Laere).
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2Trends in Plant Science, Month 2023, Vol. xx, No. xx

overcomes some of the limitations of transformation technologies with other morphogenic genes
[15,16]. The GRF4–GIF1 complex could be also expressed transiently by biolistic delivery in
wheat, and has promoted regeneration and editing efficiency in a range of cultivars [17].
Relying on the ability of short protospacer sequences (14–16 nt) to recruit transcriptional activators, a
novel approach to promote plant regeneration (termed CRISPR-Combo) has recently been pro-
posed [18]. The combination of Cas9 nuclease with regular and shorter protospacers allowed the
editing of target genes and the simultaneous activation of genes encoding developmental regulators
(WUS, WOX11, and BBM1) in poplar or rice, resulting in improved regeneration of edited plants.
Stable versus transient transformation
Genetic engineering by Rhizobium-mediated transformation mostly results in stable integration
and expression of genes. The backcrossing procedure necessary to eliminate the transgene
may present challenges, especially in heterozygous, polyploid, dioecious, or self-incompatible
crops, as well as in species with an extended juvenile period. Transformation techniques leading
to genetically modified plants (GMPs) reduce public acceptance of targeted mutagenized (edited)
plants. This makes transient expression of Cas nucleases a desirable alternative because this cre-
ates transgene-free genome-edited plant lines with reduced off-target effects. An especially ef-
fective technique in this regard is to use ribonucleoprotein complexes (RNPs) that degrade
rapidly once in the cell [19]. Polyethylene glycol (PEG)-mediated delivery of RNPs into cell wall-
free protoplasts has been accomplished in various important plant species [20–22]. To avoid
the potential toxicity of PEG in some protoplast systems, alternative transformation procedures
based on electroporation, lipofection, and biolistics can be devised [23,24]. Nevertheless, proto-
plast transformation is confronted by a series of pitfalls that limit its potential use for editing plant
genomes. Transformation success is limited by explant type and protoplast quality [25,26]. Pro-
toplasts are also very sensitive and need gentle handling. Alternative isolation systems may need
to be established, such as the 'Tape-Arabidopsis Sandwich' protoplast isolation protocol [20,27].
Regeneration relies on various parameters, including genotype, explant type, culture type, light
conditions, media composition, and oxidative stress induced during isolation and culture [28].
Furthermore, somaclonal variation may complicate the evaluation of genome-editing events.
Protoplasts are prone to these modifications, and show numerous chromosome rearrangements
in regenerated plants [29,30]. Markers linked to regeneration and/or protoclonal variation could
be designed based on chromocenter (re)assembly, reactive oxygen species (ROS) activity,
DNA methylation or hydroxymethylation, histone methylation, phytohormone ratios, or gene ex-
pression, potentially resulting in the creation of custom-made and genotype-specific regeneration
protocols that ensure the universal applicability of protoplast-based techniques.
Delivery through biolistics, leading to either stable or transient expression of editing reagents, with
different cargos and target tissues as well as editing pathways, has been reported in a range of
species [31]. Among others, transient expression of CRISPR/Cas has been achieved by
delivering DNA, RNA, or RNPs into wheat and maize [19,32,33]. In the latter species, biolistic
delivery enabled targeted mutagenesis, precise gene editing or insertion, and promoter
insertion/exchange [34,35]. Although the biolistics approach can help to overcome the limitations
of protoplast isolation and regeneration, it requires an expensive apparatus and can favor random
integration of multiple transgene copies in nuclear or organellar genomes.
Testing the functionality and efficiency of editing constructs
The efficiency of editing constructs can vary extensively. Furthermore, when several genes are
simultaneously targeted by using more than one guide RNA (gRNA), the editing efficiency of
each gRNA in the polycistronic transcripts is not the same; those located at the end of the coding
Glossary
Base editing (BE): CRISPR-based
editing technology relying on either a
Cas nickase or a dead Cas fused to a
deaminase enzyme to achieve single-
base C to T or A to G transitions.
Cas nickase (nCas9): amutant
version of Cas9 that generates single-
strand breaks in the DNA.
CRISPR/Cas: clustered regularly
interspaced short palindromic
repeats (CRISPR) are spacer
sequences that have been identified
in the genomes of prokaryotic
organisms, and are derived from DNA
fragments of infecting
bacteriophages to serve as elements
of acquired immunity to future
infections. CRISPR-associated
protein (Cas) is an enzyme that uses
CRISPR-encoded sequences as a
guide to recognize and cleave
specificstrandsofDNA
complementary to the CRISPR
sequence.
Dead Cas (dCas): also known as
endonucl ease-deficient Cas9, a mutant
form of Cas9 that has no endonuclease
activity but can bind to the DNA strand
that is targeted.
Indel: insertion and/or deletion of
nucleotide base(s) at a locus in the
genome.
Non-homologous end-joining
(NHEJ): an error-prone DNA repair
mechanism.
Prime editing (PE): an editing
technology based on a Cas nickase
fused to an engineered reverse
transcriptaseenzymeandaPE
guide RNA (pegRNA) that specifies
the target site and encodes the
desired edits.
Protospacer adjacent motif (PAM):
a short specificsequenceatthe5′or 3′
end of the target DNA sequence that is
essential for cleavage by Cas nuclease.
Site-directed nuclease type I
(SDN-1): induces Indel mutations in a
predefined region in the genome as a
result of NHEJ repair of a double-strand
break (DSB).
Site-directed nuclease type II
(SDN-2): generates specificsmall
Indel or single-nucleotide variant
(SNV) modifications as a result of
the introduction into the cell of a
repair DNA template (donor DNA)
homologous to the targeted area.
This enables precise repair of a
DSB by homologous recombination
(HR).
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sequence can have lower editing capacity [36]. Several systems have been developed to validate
editing constructs before their stable/transient transformation to produce edited tissues, including
protoplasts [19,37], hairy roots [38,39], plant cell suspension cultures (PCSCs), or a biolistics-
based leaf epidermis transient expression assay [40]. PCSCs, which reflect many physiological
and biological characteristics of a whole plant while being a stable system, allow controlled testing
of the effects of SDNs in plants. They can be utilized as a stable and uniform source of protoplasts
for transformation [20], and can be used directly for Rhizobium-mediated transformation [41]or
biolistic and electroporation-based introduction of elements for genetic engineering. A system
based on Rhizobium-mediated transformation of wheat cell suspension cultures was used to eval-
uate the editing efficiency of gRNA/Cas9 constructs designed for the ABA 8′-HYDROXYLASE 1
gene [42]. Both hairy root and PCSC systems can also be used to regenerate edited plants, par-
ticularly when other transformation methods are ineffective [43,44].
Transformation and regeneration in woody plants and polyploids
Conventional breeding of perennial woody plants presents specific challenges that include long
generation time, seasonal dormancy, huge genomes, dioecy and polyploidy, and a prolonged eval-
uation period for mature traits. All of these can be overcome by genome editing. CRISPR/Cas sys-
tems have been successfully applied in several trees such as Malus,Coffea,Citrus,andPopulus,
among others [45,46]. In woody species, however, the low transformation and in vitro regeneration
ability (except for Populus), as well as their naturally slow growth rate, represent bottlenecks for
wider implementation of genome-editing technologies [47]. In addition, chimeric regenerants, in
which only a part of the plant descends from an edited cell, may occur (e.g., in apple and pear)
[48]. Rhizobium-mediated low transformation efficiencies were increased by using other Rhizobium
species and strains [49–51], or preinfection with other bacteria such as Xanthomonas citri [52]. To
shorten the long juvenile period and promote early flowering of woody plants, thus ensuring their
early maturation, overexpression of the BpMADS4 gene may be employed [53]. Flachowsky et
al. [54] reported that overexpression of BpMADS4 in apples significantly shortened the juvenile pe-
riod and enabled early flowering. Ectopic expression of FLOWERING LOCUS T (FT)fromvarious
donor species reduced the generation time of European plum [55], Eucalyptus [56], Populus
[57], and sweet orange [58]. In addition to FT genes, arabidopsis APETALA1 (AP1) overexpression
was effective in sweet orange and citrange [59].
Polyploid plants, especially when vegetatively propagated, can present difficulties to obtain homozy-
gous individuals and can have a long reproductive cycle. These also encounter specificdifficulties for
both classical breeding and genome-editing techniques [60]. Duplicated genes, heterozygosity, re-
petitive DNA, and genome irregularities contribute to their complex genetic background. In addition,
genetic redundancy prevents functional genomic studies and breeding approaches owing to the dif-
ficulty of simultaneously mutating multiple alleles [61]. However, despite these limitations, mutations in
all alleles in hexaploid Camelina [62], tetraploid potato [63], tetraploid/hexaploid wheat [19], and other
polyploids have been obtained by using CRISPR/Casandvariousdeliverymethods. Genome editing
in polyploid plants can also be affected by poor or ineffective transfer of CRISPR/Cas reagents into
tissues with high regeneration capacity. Fluorescence-activated cell sorting (FACS) can be used to se-
lect the transformed cells. This technique, which is mostly used in mammalian and human cells, was
used for the first time in Arabidopsis [64], and was also applied successfully in some polyploid plants
such as Nicotiana benthamiana [65].
Genotype-independent in planta transformation and regeneration procedures
To overcome the bottlenecks related to de novo regeneration in vitro, genotype-independent
genome-editing protocols that do not rely on tissue culture procedures are highly desirable
in many species. However, these methods are at an advanced stage of development
Site-directed nuclease type III
(SDN-3): induces the insertion of DNA
sequences into a desired locus in the
genome enabled by the delivery of a large
(up to several kilobases) stretch of a
recombinant DNA molecule. The insertion
can take place either by HR or NHEJ.
Somaclonal variation: genetic
variation in plants regenerated through
tissue culture.
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4Trends in Plant Science, Month 2023, Vol. xx, No. xx

Key figure
Challenges and opportunities related to various Cas-based tools, their delivery into plant cells, and
the outcomes and applications
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in
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Plant
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Figure 1. Abbreviations: dCas, dead (endonuclease-deficien t) Cas; DSB, double-strand break; GE, genome editing; HR, homologous recombination; Indel, insertion /
deletion; nCas, Cas nickase; NHEJ, non-homologous end-joining; PAM, protospacer adjacent motif; pegRNA, prime editing guide RNA; RT, reverse transcriptase.
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in only a few species, and in most cases require further work to advance from the proof-of-
concept stage (Table 1). The floral dip method has been recently improved to reduce
chimerism in transformed tissues, and to enhance in planta gene targeting and frequency
of mutagenesis, but so far it has only been successfully employed to deliver editing reagents
in A. thaliana and a few close relatives [66–71]. Several studies [72–74] showed the possibil-
ity to recover CRISPR/Cas-induced mutations (edits) in non-transgenic maternal or paternal
haploids of Zea mays,A. thaliana,Triticum aestivum,andTriticum durum.However,theuse
of such an approach requires a system for haploid induction based on chromosome elimina-
tion after fertilization and the possibility to transform the haploid-inducer genotype to express
editing reagents.
In vivo delivery of editing reagents in meristems, which results in edits that are transmittable to
progeny, has been performed in only a few species [75–78]. Efficient in planta transformation
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Figure 2. CRISPR/Cas plant genome-editing (GE) methods to deliver various macromolecules into plant cells and associated challenges.
Abbreviations: GMO, genetically modified organism; gRNA, guide RNA; PEG, polyethylene glycol.
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6Trends in Plant Science, Month 2023, Vol. xx, No. xx

Table 1. Recent examples of protocols for the straightforward generation of GE plants bypassing de novo regeneration in tissue culture
a
Procedure Species Organ/tissue Editing reagents
delivered
Transfer
technology
Expression
of delivered
reagents
Target genes Edits
transmittable
to progeny
Refs
Floral dip Arabidopsis
thaliana,
Camelina sativa,
Thlaspi arvense,
Arabis alpina
Egg cells Cas9-sgRNA
Cas12a-sgRNA
plasmid DNA
Rhizobium
radiobacter
Stable AtGLABRA2,
AtALS,AtAP3,
AtLFY,AtAG,
CsFAE1,
TaFAE1,
AaSPL15
Yes [66–71]
Haploid
inducer
Zea mays,A.
thaliana,
Triticum
aestivum
Sexual
zygote,
developing
embryo
Cas9/gRNA transgenes in
pollen
None Stable
b
ZmVLHP1,
ZmVLHP2,
ZmGW2-1,
ZmGW2-2,
AtGL1,TaGT1
Yes [72]
Z. mays Sexual
zygote,
developing
embryo
Cas9/gRNA transgenes in
pollen
None Stable
b
ZmLG1,ZmUB2 Yes [73]
T. aestivum,T.
durum
Sexual
zygote,
developing
embryo
Cas9/gRNA transgenes in
pollen
None Stable
b
TaBRI1
c
,TaSD1
c
Yes [74]
Zygotes Oryza sativa In vitro
produced
zygotes
Cas9-sgRNA plasmid
DNA, Cas9-sgRNA RNP
complex
PEG-Ca
2+
Stable,
transient
DsRed2,DL,
GW7,GCS1,
PRR37
Yes [156]
Meristem
targeting
Gossypium
hirsutum
Shoot apex Cas9-sgRNA plasmid
DNA
R.
radiobacter
Stable GhCLA1,GhVP Yes [75]
T. aestivum Seed shoot
apical
meristem
Cas9-sgRNA plasmid
DNA
Biolistics Transient TaGASR7,
TaQsd1
Yes [76,78]
Solanum
tuberosum
Apical and
lateral
meristems
Cas9-sgRNA RNP
complex
Biolistics,
vacuum
infiltration
Transient StCoilin Nr
d
[77]
De novo
induction of
meristems
Nicotiana
benthamiana
(Cas9
+
)
Cotyledons
(seedlings),
cut shoot
apices (adult
plants)
Plasmid DNA [sgRNA +
developmental regulators
(DRs)
e
]
R.
radiobacter
Stable,
transient
NbPDS1,
NbPDS2
Yes [79]
Virus-induced
genome
editing (VIGE)
N. benthamiana
(Cas9
+
)
Leaves sgRNA::FTmRNA
sgRNA::
tRNA
Me
/tRNA
Gly
/tRNA
Ile
[tobacco rattle virus
(TRV)-based vectors]
R.
radiobacter
Transient NbPDS,NbAG Yes [84]
N. benthamiana
(Cas9
+
)
Leaves sgRNA::tFTmRNA [potato
virus X (PVX) vector]
R.
radiobacter
Transient NbFT,NbPDS3,
NbXT2B
Yes [157]
A. thaliana
(Cas9
+
)
Leaves sgRNA::tFTmRNA [cotton
leaf crumple virus (CLCrV)
vector]
R.
radiobacter
Transient AtGL2,AtBRI1 Yes [158]
T. aestivum
(Cas9
+
)
Leaves sgRNA with/without
fusion to FT or tRNA
mobile elements [barley
stripe mosaic virus
(BSMV) vectors]
None Transient TaPDS,TaGW2,
TaGASR7
Yes [159]
N. benthamiana Leaves Cas9-sgRNA [Sonchus
yellow net rhabdovirus
(SYNV) vector]
R.
radiobacter
Transient GFP transgene,
NbPDS,
NbRDR6,
NbSGS3
No
f
[81]
(continued on next page)
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protocols require further development. Gene-edited N. benthamiana plants have been recently
obtained by in situ delivery of single guide RNA (sgRNA) and several combinations of develop-
mental regulators which induced the formation of new meristems on somatic tissues in vivo
[79]. The latter study made use of transgenic plants expressing Cas9, and the feasibility of
codelivering Cas nuclease, sgRNA, and developmental regulators remains to be demonstrated.
In addition, to avoid negative pleiotropic effects, the expression of developmental regulators
should be regulated.
Several viruses have been considered as vectors for delivering editing reagents in planta,but
their limited ability to accommodate large molecules (e.g., Cas9) and/or infect the germline
prevents the efficient induction of edits and their transmission to progeny [80]. Recently, the
exploitation of new viruses made it possible to overcome the first limitation [81–83], and fusion
of sgRNA to meristem-specific regulatory elements increased the efficiency of mutation
transmission through generations [84]. A combination of the two approaches and/or the use
of smaller nucleases [85] might open new perspectives for alternative genome-editing
methods. Nanoparticles have been also suggested as novel delivery carriers for tissue
culture-free genome editing [86] and pollen engineering [87,88],buteveninthiscasethesize
of Cas9 and respective nucleases presently limits their delivery through the cell wall and
subsequent editing of the germline.
Transgene-free heritable editing was recently accomplished in Arabidopsis by transforming
rootstocks with Cas9 and gRNA sequences to which tRNA-like motifs had been added [89].
As a result, mobile editing reagent transcripts could move from the rootstock to the scion,
thus allowing germline editing. Alternative ways to deliver and express mobile editing reagents
in the rootstock may allow this novel approach to be exploited in various combinations of
compatible grafts.
Mutant detection
To detect edits after CRISPR/Cas-induced genomic modifications, several detection methods
can be applied. Because different methods have distinct outcomes, it is not reliable to compare
studies regarding the efficiency of mutation induction. To make studies comparable, standardiza-
tion will be essential to determine/quantify the mutation efficiency and identify which standard
Table 1. (continued)
Procedure Species Organ/tissue Editing reagents
delivered
Transfer
technology
Expression
of delivered
reagents
Target genes Edits
transmittable
to progeny
Refs
N. benthamiana Leaves Cas9-sgRNA [foxtail
mosaic virus (FoMV)
vector]
R.
radiobacter
Transient NbPDS No [82]
Nanoparticles T. aestivum Leaves Cas9-sgRNA plasmid
DNA (carbon dots)
Foliar
spraying
Transient SPO11 No [160]
Grafting A. thaliana Egg cells
(rootstock)
Cas9-TLS
g
, sgRNA-TLS
plasmid DNA
R.
radiobacter
Stable (in
rootstock)
AtNIA1 Yes [89]
a
In some cases, only in vitro culture of pre-formed meristems was carried out.
b
In haploid-inducer lines.
c
Conserved across all two (AABB) or three (AABBDD) homeologs of the target genes in durum and bread wheat, respectively.
d
Not relevant.
e
Developmental regulators (various combinations of BBM,Wus2,ipt,STM).
f
Edits were recovered only in the progenies of plants regenerated in vitro.
g
TLS, tRNA-like sequence motifs.
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8Trends in Plant Science, Month 2023, Vol. xx, No. xx

data should be obtained as an outcome. Regardless of the detection method, specificity regard-
ing the exclusive response of the target of interest is a key property. Which technique is suitable
for a specific application largely depends on the data required; for example, whether it is
necessary to determine the exact alteration, and what knowledge is available about the target
sequence, gene, and gene family (Figure 3). Mutational outcomes are not random because
they depend on the DNA sequence at the targeted location [90]. Data from more than 10
9
mutational outcomes revealed that most mutationsareinsertionsofasinglebasepair,short
deletions, or longer micro-homology-mediated deletions [90]. This presents a challenge in devel-
oping analytical methodologies that can identify unique markers of such small genetic alterations.
PCR-based methods
Real-time PCR methods are often used to detect genome-edited plants, but they have limita-
tions related to the detection of single-nucleotide variants (SNVs). A crucial aspect here is the
conjunction of real-time PCR with locked nucleic acids (LNAs) in primer design because this
increases assay specificity in genome-edited plants such as in canola [91]andrice[92].
Trends
Trends
in
in
Plant
Plant
Science
Science
Figure 3. Detection methods used in the development of protocols for CRISPR/Cas genome editing of plants. Abbreviations: ddPCR, droplet digital PCR;
DSDecode, degenerate sequence decode; ICE, inference of CRISPR edits; HRM, high-resolution melting; Indel, insertion/deletion; NGS, next-generation sequencing;
qPCR, quantitative PCR; RFLP, restriction fragment length polymorphism; SDN, site-directed nuclease; SNV, single-nucleotidevariant;TIDE,tracking of indels by
decomposition; WGS, whole-genome sequencing.
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Droplet digital PCR (ddPCR) [5,93] is a promising tool for genome-editing detection, as has
been shown for detecting SNVs in oncology research [94], in edited cells [95,96], and recently
in edited plants to identify wild-type, homozygous, and heterozygous mutations induced by
CRISPR/Cas9 in rice [92]andrapeseed[93]. Similarly, a duplex ddPCR approach distin-
guished CRISPR/Cas-induced small Indels in ALPHA-GLIADIN from large deletions in poly-
ploid bread wheat [97]. Furthermore, high-resolution melting (HRM) analysis is a post-PCR
approach based on monitoring the gradual denaturation of amplicons that can detect small
sequence differences [98] such as small Indels or even SNVs in gene-edited rice [99]. How-
ever, HRM analysis also has limitations associated with the use of small target amplicons
(100–200 bp) which might hamper the detection of large Indels. All these PCR-based tech-
niques are relatively cheap, easy to implement, and allow rapid routine screening of gene-
edited mutant collections. However, they only indirectly show the presence of a mutation,
and do not identify the exact alteration at the nucleotide level.
Sequencing-based methods
Amplification and sequencing of the target loci allow identification of the specific nucleotides that
are deleted, inserted, or substituted. In addition, when a large number of regenerated polyploid
plants need to be analyzed for the presence of the multi-allelic structure, sequencing-based de-
tection methods are valuable. Furthermore, many traits are determined by multiple small-effect
genes operating in gene families and/or complex interactive networks, and thus require high-
throughput, multiplex CRISPR approaches. Off-targets also need to be determined. All of these
represent additional challenges and increase the demand for efficient detection techniques for
proper screening and data analysis based on new sequencing-based and bioinformatic tools.
Sanger sequencing of (cloned) amplicons enables the identification of induced mutations at on-
target sites and makes it possible to determine the frequency and the type of mutations at a spe-
cific locus. However, Sanger sequencing can lead to confusing results when applied to polyploid
organisms with heterozygous or multiallelic mutations [100]. Bioinformatic tools such as TIDE
(tracking of indels by decomposition; http://tide.nki.nl)andICE(inferenceofCRISPRedits;
https://ice.synthego.com/#/) can be used for automatic decoding of overlapping electrophero-
grams derived from PCR amplicons that hold different types of mutations. The sensitivity of
Sanger sequencing is (only) about 15%, meaning that if a pooled strategy is used, 15% of the
pool must contain the mutation before it can be detected. Low-efficiency editing is thus likely to
be overlooked by Sanger sequencing [101].
Recent advances in sequencing technologies have led to the development of high-throughput
next-generation sequencing (NGS) technologies using second- (short-read) and third-genera-
tion (long-read) sequencing technologies. These enable massively parallel sequencing and
analysis of heterogeneous samples. NGS sequencing has a sensitivity of 0.1% to 1%, allowing
efficient pooling and screening of protoplast samples. To analyze the resulting huge datasets,
bioinformatic tools are available such as CRISPResso2 [102] and SMAP (stacked mapping an-
chor points) [103]. In the study of Lorenzo et al. [1], multiplex genome editing of whole gene
families was combined with crossing schemes to improve complex traits (yield, drought stress).
NGS sequencing and SMAP data analysis were used to screen for knockouts of 48 growth-
related genes in maize, and a collection of >1000 gene-edited plants were regenerated. Recent
progress is also reported related to gRNA library-based CRISPR screens for high-throughput
loss-of-function screens in plants, even at a genome-wide scale [104,105]. gRNA libraries can
be created and introduced in bulk in plant cells in a way that individual cells receive different
gRNAs. Unique gRNAs can serve as barcodes and then be identified in a pool of cells by
high-throughput sequencing. Such CRISPR screens enable rapid connection between the ge-
notype and the phenotype.
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In addition to targeted sequencing, NGS also enables whole-exome and whole-genome se-
quencing (WGS) which is an untargeted detection approach for unknown alterations (small Indels
and SNPs) as well as a way to detect structural variants such as inversions, rearrangements, du-
plications, and major deletions [106,107]. WGS therefore makes it possible to detect on-target
mutations induced by Cas endonucleases including off-target mutations induced by endonucle-
ases as well as naturally occurring mutations. WGS has demonstrated its efficacy for revealing
rare off-target mutations in rice [108], cotton [109], and grapevine [110].
In general, NGS is currently considered to be the most comprehensive and reliable technique for
analysis of genome-editing events [111], even in polyploid or chimeric plants, as well as for tracing
large insertions/deletions and mutations in multi-edited plants. Knowing the exact mutation at the
nucleotide level allows the mutated allele to be placed in its gene context and thus enables the
effect of the mutation on the structure and function of the encoded protein to be predicted. On
the other hand, NGS produces large datasets. However, becausehigh-throughput multiplex mu-
tation screening is possible, the per sample per locus costs are substantially reduced. In addition,
although datasets can be huge, especially for plants with large genomes, they are systematically
structured such that high-throughput automated analysis becomes possible. Skilled investiga-
tors and suitable bioinformatic pipelines are able to process and detect all haplotypes, at all
loci, in all samples, and in parallel. However, even using NGS, it is impossible to distinguish an ar-
tificially edited genome from one containing natural mutations.
New CRISPR/Cas actions and platforms
Gene regulation
CRISPR/Cas can be employed in several ways to regulate gene expression. miRNAs are en-
dogenous small RNA molecules that control the abundance of target mRNA in diverse path-
ways [112]. Fine-tuning of their abundance by CRISPR/Cas to regulate miRNA-regulated
plant processes is feasible; however, the research is in its early phase and still represents a
challenge, especially in polyploid species [112]. One drawback is that miRNA editing with a
sgRNA is not necessarily efficient because frameshifts do not always affect miRNA function
[113]. For this reason, dual sgRNA design is recommended to target both ends of a mature
miRNA coding sequence. In an optimal situation, this would result in the removal of the
whole miRNA sequence, or at least sufficiently perturb the structure of pre-miRNA to impair
the processing of miRNA [114]. miRNA modulation by a sgRNA is also feasible, although
less efficient [115,116].
Different approaches have been implemented to target miRNA genes in several plant species (Ta-
ble S2 in the supplemental information online). miRNA control of gene expression can also be al-
tered by inducing mutations in miRNA binding sites of regulated genes.
In addition, cis-regulatory elements (CREs) in the genes to be modified can be disrupted or re-
placed by CRISPR/Cas-based genome editing [34,117]. In tomato, the use of CRISPR/Cas to
dissect CREs allowed the generation of novel variation underlying quantitative trait variation and
pleiotropy that both control the phenotypic variation of useful traits [118–120]. Finally, gene ex-
pression can also be modulated, without stable genome modification, by fusing dCas9 to tran-
scriptional effectors or epigenetic modulators, or by exploiting the cited ability of short
protospacers, combined with a standard nuclease, to recruit transcriptional activators
[18,121,122]. Challenges for future research on dCas-based technologies include improvement
of the activation/repression efficiency and of the epigenetic manipulation specificity, together with
the possibility of controlling gene expression simultaneously in multiple targets and/or in an induc-
ible or tissue-specific manner [121].
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Advanced CRISPR/Cas variants
Despite many successful examples of first-generation CRISPR methods in plants, introduced
changes were predominantly knockout mutations of the targeted loci. Advanced plant breed-
ing programs need precise changes in plant genomes, which cannot be accomplished by
non-homologous end-joining (NHEJ) following the generation of a DSB. Moreover, many
loci cannot be targeted by the first generation of Cas9 nucleases from Streptococcus pyogenes
(SpCas9) owing to the lack of suitable protospacer adjacent motif (PAM) sequences.
Advanced CRISPR/Cas variants are therefore being developed [123] for precise targeting using
newly discovered SDN or modified Cas proteins combined with the catalytic activity of DNA-
modifying proteins. Acquired fusion proteins for BE and PE can generate single-nucleotide sub-
stitutions, in-frame insertions, and large deletions. In addition, these variants allow DSB-free and
template-free editing with improved efficiency and increased specificity. Some of the new variants
have already been successfully used in plants (Table 2). Although these new techniques will need
more experimental data to be widely and routinely applied for crop improvement, they provide a
more efficient induction of intended edits, thus increasing the chance of obtaining the desired mu-
tants even from plant genotypes with low regeneration capacity. Wider implementation of these
new CRISPR variants in different plant species and their optimization for plant expression
(e.g., Golden Gate Domestication, codon optimization) will foster plant genome editing. The
new CRISPR toolboxes are already overcoming bottlenecks related to PAM restriction of tradi-
tional SpCas9 and target specificity. The large size of SpCas9 is addressed by the adoption of
smaller SDN enzymes [124–126] that, combined with split-Cas systems [127,128], will also en-
able in planta virus-mediated expression of SDN.
Opportunities and challenges related to specific applications
Biotic and abiotic stresses
CRISPR/Cas genome editing has been applied to enhance disease resistance and abiotic stress
tolerance. Despite the great potential and some outstanding results [129], several issues still limit
its efficiency as a tool for mitigation of plant stresses.
To induce disease resistance via genome editing, the identification and functional annotation of
plant susceptibility (S) and/or resistance (R) genes is needed. This is labor-intensive and demands
access to whole-genome sequences. However, limited or no data are available on the molecular
functions of S/Rgenes in most non-model plants [130]. Furthermore, many Sgenes have dual
roles in plant physiology and susceptibility to pathogens, which often makes them essential for
the survival of the host. Inactivating these genes often leads to disease resistance but is also ac-
companied by pleiotropic effects including plant growth inhibition, phenotypic abnormalities, and
increased susceptibility to abiotic stress and/or other pathogenic agents [131–134].
Similarly to other methods for inducing disease resistance, the widespread and long-term plant-
ing of gene edited plants, especially in monocultures, might lead to the appearance of new or
adapted pathogen strains. Simultaneous protection against different races or strains of the
same species and/or several taxonomically unrelated pathogens can be an additional challenge
[130,135,136]. The possibility of using CRISPR/Cas for the fast induction of new mutations
that lead to plant resistance can help to manage resistance genes and edited plants such as
by producing multi-lines or stacked lines with multiple resistance mechanisms.
The frequency and intensity of climate extremes are increasing, which means that disease resis-
tance should be combined with tolerance to abiotic stress. Resilience in the face of abiotic stress
usually depends on complex morphophysiological mechanisms controlled by multiple genes,
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Table 2. New CRISPR/Cas variants for enhanced and more precise plant genome editing
a,b
CRISPR variant Component Novelty or mode of action Expression platform and editing
efficiency
Refs
CBE (cytosine
base editor)
nSpCas9
a
+ rAPOBEC1 (rat cytidine
deaminase) + UGI (uracil DNA
glycosylase inhibitor)
Introduction of nucleotide changes
without inducing a DSB: C to T base
transition.
Higher efficiency compared to
HDR-mediated base pair substitution,
lower occurrence of undesirable
mutations.
Rhizobium radiobacter-mediated
transformation of watermelon and
cauliflower.
Efficiency: 23% in watermelon T0
generation and 22% or 87% in
cauliflower, depending on the targeted
locus.
[161,162]
nSpCas9 or SpRY (PAM-less) + UNG
(uracil DNA glycosylase) + rAPOBEC1
Introduction of nucleotide changes
without inducing a DSB: C to G base
transversion.
Higher efficiency than HDR-mediated
base pair substitution, lower frequency
of undesirable mutations.
Rice and tomato protoplasts; R.
radiobacter-mediated transformation of
rice and poplar.
Efficiency: C to G substitution in 38% of
transgenic rice lines and 6.3% in poplar;
C to T edits and Indels among
byproducts.
[163]
nSpCas9(D10A) + rationally designed
human A3Bctd-BE3
Reduced sgRNA-independent genomic
off-target activity of CBEs producing
mainly single and double C to T edits,
with marginally reduced on-target
activity in rice.
Rice protoplasts transfection and R.
radiobacter-mediated transformation of
rice calli.
Efficiency: up to 30% on-target effi-
ciency across the four target sites and
reduced sgRNA-independent off-target
activity.
[164]
nSpCas9-NG + evoFERNY + rice UNG
or human UNG
Introduction of nucleotide changes
without inducing a DSB.
Recognition of NG PAM sequence.
C to T base transition, C to G or C to A
transversion.
Efficient monoallelic base editing with
the significant number of
insertion/deletion byproducts.
R. radiobacter-mediated transformation
of rice calli.
Efficiency: Up to 21% of C to T, up to
8.2% of C to G and up to 3.7% C to A
on-target efficiency in T0 transformants.
[165]
nSpCas9-pBE and VQRn-pBE Petromyzon marinus cytidine
deaminase 1 (PmCDA1) and uracil DNA
glycosylase inhibitor (UGI) fused to
SpCas9. VQRn variant created by
D1135V, R1335Q, and T1337R amino
acid substitutions in the SpCas9
sequence. Efficiency additionally
increased by modified gRNA.
C to T base substitutions.
Deamination window within 1–7nt
upstream the PAM.
R. radiobacter-mediated transformation
of rice.
Efficiency: up to 90% in rice T0 plants.
[166]
ABE (adenine
base editor)
nSpCas9 (D10A) + engineered E. coli
tRNA adenine deaminase (TadA)
Introduction of nucleotide changes
without inducing a DSB.
A to G base transition.
Higher efficiency than HDR-mediated
base pair substitution, lower occurrence
of undesirable mutations.
R. radiobacter-mediated transformation
of rice.
Efficiency: A to G transition in 26% of
transgenic lines.
[167]
SpGn + adenine deaminase TadA8e
with E. coli endonuclease V (EndoV) or
human alkyladenine DNA glycosylase
(hAAG)
Introduction of nucleotide changes
without inducing a DSB.
A to G base transition.
High efficiency and broad target range
owing to the variable PAM sequence.
R. radiobacter-mediated transformation
of rice calli.
Efficiency: average 56% of A to G
on-target efficiency.
[165]
PhieABE variants: nSpCas9-NG,
nSpGornSpRY (PAM-less) + TadA8e
adenine deaminase + single stranded
DNA-binding domain (DBD)
Introduction of nucleotide changes
without inducing a DSB.
A to G base transition at the NGN PAM
target site.
DBD enhanced on-target activity and
decreased off-target and self-editing.
Rice T0 transformation with R.
radiobacter.
Efficiency: up to 78.5% average
on-target editing across the 17 target
sites.
[168]
(continued on next page)
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Table 2. (continued)
CRISPR variant Component Novelty or mode of action Expression platform and editing
efficiency
Refs
STEME (saturated
targeted
endogenous
mutagenesis
editors)
nSpCas9 (D10A) + cytidine deaminase
+ adenosine deaminase + UGI
Saturating mutagenesis of a single locus
with a single protein that uses a single
sgRNA to achieve C:G > T:A and A:T >
G:C substitutions.
PEG-mediated transfection of rice
protoplasts.
Efficiency: STEME-1 edited cytosine
and adenine at the same target site with
C>Tefficiency up to 61.6% and
simultaneous C > T and A > G efficiency
up to 15.1%. STEME-NG produced
73.2% mutagenesis.
[169]
SWISS (CRISPR
simultaneous and
wide-editing
induced by a
single system)
nSpCas9 (D10A) + sgRNA scaffolds
with different RNA aptamers + binding
proteins fused with cytidine or
adenosine deaminase
Multiplex base editing, insertions and
deletions.
Editing adenosines and cytidines at
separate sites.
R. radiobacter-mediated transformation
of rice callus cells.
Efficiency: cytosine conversion 25.5%,
adenine conversion 16.4%, Indels
52.7%, and simultaneous triple
mutations 7.3% in rice mutants.
[170]
Prime editing –
PE, PE2, PE3
PE: nSpCas9 (H840A) + RT + pegRNA
PE3: PE, pegRNA and a second gRNA
nicking the non-edited DNA strand to
trigger repair of the edited strand
PE3b: PE, pegRNA and a second
gRNA nicking the non-edited DNA
strand with a spacer matching the
edited sequence
Reverse transcription of an editing
template from pegRNA (a modified
gRNA) directly into the target locus
which is nicked and extended by the
prime editor.
Introduction of single or multiple base
substitutions, Indels, long deletions.
Maize inbred lines transformation with
R. radiobacter LBA4404/pVIS1–VIR2.
Rice protoplasts are transfected by the
PEG-mediated method.
Efficiency: increased efficiency of PE in
maize from 6.5% up to 71.7% by two
strategies: the Csy4 RNA processing
system and the tRNA and HDV
ribozyme RNA-processing system
integrated with two drivers,
polymerase II (35S
enhancer-CmYLCV) and III (shortened
U6-26) promoters. From 1% to 7%
efficiency in rice protoplasts depending
on the vector used.
[171]
ePPE Based on original nSpCas9 + M-MLV
RT. The RNAse H domain was removed
from fused M-MLV RT and additionally
fused to a viral NC peptide with nucleic
acid chaperone activity.
Rice protoplasts and transformed
plants.
Efficiency: 5.8-fold higher than the
original plant PE.
[172]
enpPE2 nSpCas9 with sequence modifications
(R221K/N394K; PEmax) fused to
M-MLV RT plus modified pegRNA
expressed under a composite CaMV
35S enhancer-CmYLCV-U6 promoter.
Rice protoplasts and transformed
plants.
Efficiency: 64.58–77.08% in rice T0
plants.
[173]
AFIDs
(APOBEC–Cas9
fusion-induced
deletion systems)
SpCas9 + human APOBEC3A (A3A) +
uracil DNA-glucosidase + apurinic or
apyrimidinic site lyase
Introduction of larger deletions. PE G-mediated transfection of rice and
wheat protoplasts, R.
radiobacter-mediated transformation
of rice callus cells, and biolistic trans-
formation of immature wheat embryo
cells.
Efficiency: AFID-3 generated deletions
from 5′-deaminated C bases to the
Cas9-cleavage site in rice and wheat
protoplasts. One-third of these dele-
tions in protoplasts (30.2%) and
regenerated plants (34.8%) were
predictable.
[174]
Cas9-VirD2 SpCas9 + VirD2 relaxes protein from
R. radiobacter to tether end-protected
single-stranded DNA repair template
for HDR to the targeted DSB
More efficient gene targeting through
HDR.
Bombardment of rice calli with
plasmids.
Efficiency: fivefold increase over the
non-tethered control in rice. Up to 9.9%
efficiency of gene editing through HDR
in the absence of selection.
[175]
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and thus implies the need to develop multiplex CRISPR-based approaches [130,135]. The role of
tolerance (T) and sensitivity (S) genes, which positively or negatively regulate stress tolerance and
adaptation, has been proposed [137]. Genes having a structural or a regulatory role, as well as
cis-regulatory sequences, can be edited by CRISPR/Cas, but the possible occurrence of detri-
mental pleiotropic effects after such manipulations is an additional challenge to consider. In addi-
tion, modification of R/Tgenes requires the development of alternative, and often more complex,
approaches based on genome editing/replacement by homologous recombination (HR), BE, or
PE. The strategy of Lorenzo et al. [1] is based on the combination of simultaneous editing of
gene families using up to 12 sgRNAs followed by subsequent crossings. This resulted in multiple
mutant maize plants with improved response to water-deficient conditions which could be utilized
in breeding programs.
Field trials with genome-edited plants are of paramount importance to confirm the efficacy of in-
duced changes under normal production conditions, to demonstrate the durability of resistance
or tolerance, and to check for the absence of off-target effects.
De novo domestication and rewilding
In the current scenario of global population increase and global climate changes, CRISPR/Cas
approaches can facilitate the development of novel plant varieties that combine good agronomic
performance with adaptability to stress-inducing environments and low-input management prac-
tices. This has the potential to enhance plant genetic resources for food and agriculture (PGRFA)
and their utilization in agricultural systems based on 'sustainable intensification'. Although plant
Table 2. (continued)
CRISPR variant Component Novelty or mode of action Expression platform and editing
efficiency
Refs
CRISPR PLUS:
C9R and C9G
C9R: SpCas9 + 5′-to-3′exonuclease
RecJ
C9G: SpCas9 + gfp
Increase in both mutagenesis and
knock-in efficiency; off-target effects
were not significantly increased relative
to the Cas9 (C9) structure alone.
PEG transfection of Nicotiana
benthamiana protoplasts with RNPs.
Efficiency: 2.6-fold increased editing
efficiency of C9R compared to control
C9 in protoplasts of N. benthamiana.
[176]
CRISPR-Combo Cas9-Act3.0 system: catalytically
active Cas9 nuclease, MS2
bacteriophage coat protein
(MCP)-SunTag-activator complex, and
a 15 or 20 nt sgRNA
Gene editing or gene activation
optimization.
Arabidopsis plants, poplar and rice calli,
and rice and tomato protoplasts.
[18]
CasΦCasΦvariants: wtCasΦ, vCasΦ, and
nCasΦ
Small, hypercompact enzyme, requiring
a T-rich minimal PAM, efficient over a
wide range of working temperatures,
sensitive to chromatin environment, and
with higher editing specificity.
Arabidopsis and maize protoplasts, and
transgenic Arabidopsis plants.
[125,177]
Cas12j2 variants: wtCas12j2,
vCas12j2 and nCas12j2
Small enzyme, highly specific nuclease
activity, T-rich PAM site (preferably
5′-NTTV-3′). Efficient editing in
non-dividing cells.
Gene activation and epigenome editing
for fine-tuning target gene expression in
plants.
Rice protoplasts and stable transformed
lines, tomato protoplasts, and poplar
transgenic plants.
[126]
a
Abbreviations: CaMV, cauliflower mosaic virus; CBE, cytosine base editor; CmYLCV, Cestrum yellow leaf curling virus; DSB, double-strand break; evoFERNY, evolved
cytosine deaminase; HDR, homology-directed repair; HDV, hepatitis delta virus; M-MLV, Moloney murine leukemia virus; NC, nucleocapsid; PAM, protospacer adjacent
motif; PEG, polyethylene glycol; pegRNA, prime editing guide RNA; RNP, ribonucleoprotein; RT, reverse transcriptase.
b
nSpCas9, SpCas9 nickase from Streptococcus pyogenes in which one endonuclease domain is mutated (D10A or H840A) and therefore only introduces single-strand
nicks.
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domestication and genetic improvement have contributed to the present level of productivity and
quality traits in elite cultivars when grown under favorable environments, these also have reduced
genetic diversity and less resilience to stress conditions in comparison to their wild relatives. This
has been confirmed at the genomic level by de novo assembly of the pan-genome in several
crops, which highlighted the evolution of copy number and the presence/absence of variants.
In some cases these were also related to response to stress conditions [138–140].
To capture this 'lost diversity' in elite cultivars, the 'rewilding' concept has been advanced by
Palmgren et al. [141]. It aims to reintroduce mutations that are no longer present in the culti-
vated gene pool but are still available in wild progenitors (Figure 4). Rewilding can be achieved
by introgression breeding, specific insertion of candidate genes, and precision mutagenesis.
CRISPR/Cas-based approaches, including more advanced technological developments
(e.g., multiplex methods, BE, and PE), can help to achieve these goals by favoring not only
gene knockout but also gene knock-in and recombination at specified genomic locations [142].
To combine agronomically desirable traits with useful traits from wild species (Figure 4), de novo
domestication approaches, including redomestication of crop progenitors as well as domestica-
tion of wild species, have been demonstrated in Solanum pimpinellifolium (a stress-tolerant wild
tomato relative), Physalis pruinosa (groundcherry, an orphan crop distantly related to tomato),
and Oryza alta (a wild tetraploid rice) by modifying 'domestication genes' via CRISPR/Cas-
based technologies [138,143–145]. Because regulatory variants have been linked to domestica-
tion traits in various crops [146,147], novel de novo domestication approaches will soon involve
CRISPR/Cas approaches in the modification of cis-regulatory sequences [142,148]. Other wild
species, in some cases relatives of important crops such as potato, kiwi, and pepper, are in
the pipeline for further applications [149,150].
Landraces derived from domestication and participatory breeding show adaptation to regional
growing conditions and are appreciated by local consumers. Nevertheless, they often present
defects that are incompatible with modern agriculture. CRISPR/Cas methods can be used to
correct such defects without altering the genetic background and the general phenotype
(Figure 4).
Nevertheless, to fully exploit these new breeding approaches, it will not only be important to apply
available editing tools but also to select suitable genetic materials to start with, as well as to de-
velop high-quality genomic information and efficient methods for the delivery of editing compo-
nents and regeneration of edited cells.
Concluding remarks and future perspectives
In the first decade of use, CRISPR/Cas technology exceeded initial expectations by providing a
precise, easy to implement, and high-throughput tool for targeted genetic modifications to
plant research and breeding activities [151]. It was quickly adopted in most life-science laborato-
ries, even those with no previous expertise of genetic modifications. Owing to its broad range of
applications and adaptability to various molecular biology protocols, CRISPR/Cas is now the
method of choice in fundamental research. It is used predominantly for gene targeting and knock-
outs, regulation of gene expression, introduction of heterologous DNA sequences at predefined
genomic loci, and chromosome tagging. The future of plant genome editing relies on an expan-
sion of the use of CRISPR/Cas based on more efficient multiplexing, high-throughput editing
strategies, and applications towards chromosomal rearrangements and epigenomic changes.
Furthermore, current difficulties with delivering Cas nucleases and gRNAs into plant organelles
hinder their use for editing plastomes and chondriomes.
Outstanding questions
Is CRISPR/Cas technology readily
adoptable by a pplied plant br eeding
programs? The tissue culture
response of crop geno types is one of
the main bottlenecks for implementing
CRISPR/Cas-based genome editing in
applied breeding programs. More re-
search will be necessary to develop
novel transient methods for delivering
editing reagents into plant cells. Such
methods are unlikely to be based on
conventional tissue cultureregeneration
protocols.
Can complex agronomic traits, such as
productivity or tolerance to stress, be
efficiently modified by CRISPR/Cas-
based technologies? Functional studies
and breeding of m ultigenic trait s will r e-
quire the development of efficient high-
throughput and multiplexed CRISPR
methods.
Are techniques available to detect
products derived from CRISPR/Cas-
based editing? Several advances
have been proposed to trace gene-
edited plants based on ddPCR and
NGS. However, these techniques do
not distinguish edited genomes from
naturally occurring mutations. This is a
major bottleneck for the EU approval
of gene-edited plants.
Are available CRISPR/Cas-based
editing tools sufficient to deal with all
intended applications? Several new
platforms have been developed in the
past decade. Nevertheless, the dis-
covery and development of new Cas
enzymes characterized by small size,
high fidelity, and different sequence
recognition signals will be necessary
to develop novel delivery methods, re-
duce off-target mutations, and
broaden the sequence typ es that can
be modified.
Trends in Plant Science
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16 Trends in Plant Science, Month 2023, Vol. xx, No. xx

Trends
Trends
in
in
Plant
Plant
Science
Science
Figure 4. Application of CRISPR/Cas to induce mutations aiming at de novo domestication of crop wild
relatives, rewilding of elite varieties, or correction of defects in landraces. Abbreviation: CWR, crop wild relative.
Trends in Plant Science OPEN ACCESS
Trends in Plant Science, Month 2023, Vol. xx, No. xx 17

The application of high-throughput screening in plants is still in its infancy compared to animal cell
culture systems. Nevertheless, high-throughput CRISPR experiments will be expanded [152], en-
abling the generation of mutant collections and CRISPR libraries using multiplexed CRISPR
vectors and combinatorial vector assembly. New techniques for pooled plant transformation
and CRISPR knockout screens will be developed. In addition, editing efficiencies are and will
be increased by changing promoters, using tissue-specific promoters, and Cas codon optimiza-
tion. New Cas enzymes will be discovered or are being developed, thus minimizing the limits on
choosing the right target.
When performing extensive CRISPR screens in plants, it is crucial to produce a large number of
regenerated plants. When studying crops with low transformation efficiency (e.g., maize), only a
few genotypes will have the capacity to generate large screening populations. Improving the ef-
ficiency of plant regeneration will make it possible to scale up CRISPR studies. Alternative
methods [virus-induced genome editing (VIGE) and nanotechnology-based] have been devel-
oped to avoid the need for de novo regeneration from tissue culture. However, to increase the
adoption of these technologies, it will be important to overcome the limitations set by the size
of the Cas enzyme.
In the future it will be important to broaden the targets for crop improvement. Researchers and
breeders need to have a better understanding of the biological processes and genes involved,
as well as of the pathways and their interaction with environmental factors. CRISPR will be a
very valuable tool for functional gene studies. Genome-scale CRISPR mutant libraries could be
made if some limitations are overcome [152], namely by developing prediction tools for gRNA ef-
ficiency and the editing outcome, recording genome-editing events at the single-cell level, and
setting up large-scale combinatorial CRISPR screens.
Regarding the implementation of CRISPR-based systems in plant breeding schemes, the depen-
dence on genotype for in vitro regeneration capacity undoubtedly remains one of the main chal-
lenges. However, some strategies discussed here open new perspectives, such as the activation
of endogenous genes encoding developmental regulators, (transient) coexpression of
transgenes with the same function, and the development of in planta editing protocols. The use
of CRISPR-Combo to activate flowering-promoting factors [18] can also play a game-changing
role in reducing juvenility in woody plants and shortening the duration of breeding cycles. Finally,
analyses of pan-genomes have revealed the widespread presence of natural structural variants,
highlighting their role during the evolution and domestication of crop plants. The possibility of in-
ducing targeted chromosomal rearrangements sets new horizons for the increase/decrease of
meiotic recombination, resulting in wider access to genetic variation in crop relatives in the former
case and preservation of positive gene assets in the latter [153–155].
The technical challenges of CRISPR/Cas have been either (partially) overcome or will be tackled in
future research, but the uncertain regulatory status of genome-edited plant varieties in the EU re-
mains a major limitation for the applicability of CRISPR/Cas and other genome-editing technolo-
gies in plant breeding. Currently, EU legislation hinders adapted crops from being planted in the
field and/or being marketed. The current regulation of genome-edited organisms as genetically
manipulated organisms (GMOs), regardless of the type of introduced changes, restricts the use
of precise plant breeding by genome editing to major, broad-acre crops such as maize and
soybean. Their cultivation is thus financially feasible only for large (multinational) companies.
To address these challenges in the EU, various initiatives [Cooperation in Science and
Technology (COST) Action PlantEd 2019–2023, https://plantgenomeediting.eu/; and EU-SAGE
(Sustainable Agriculture through Genome Editing), https://www.eu-sage.eu/] have been
Trends in Plant Science
OPEN ACCESS
18 Trends in Plant Science, Month 2023, Vol. xx, No. xx

launched. These initiatives aim to disseminate science-based information about new breeding
techniques, which will be necessary to foster innovation and support advanced plant breeding
as part of the transition to a more sustainable food production system in a fast-changing and
challenging environment. New data on plant genome editing should promote the development
of European and EU Member State policies based on scientific and socioeconomic aspects,
thus enabling responsible use of genome editing for sustainable agriculture and food production
(see also Outstanding questions).
Acknowledgments
This article/publication is based upon work from COST Action PlantEd (CA18111), supported by COST (European Cooper-
ation in Science and Technology) (www.cost.eu). The authors thank Gaetano Guarino for help with the artwork and Miriam
Levenson for text editing of the manuscript.
Declaration of interests
The authors declare no conflicts of interest.
Supplemental information
Supplemental information associated with this article can be found, in the online version, at https://doi.org/10.1016/j.tplants.
2023.05.012.
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